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Translational quality control mechanisms that mitigate stop codon readthrough and ensure protein homeostasis
Get PDFCells invest tremendously to maintain the fidelity for transcription and translation to ensure accurate transmission of the genetic code into proteins. Yet, errors may occur at each stage. During transcription, errors arise at a rate of ~10-5-10-4 per base, whereas the error rate during translation is around a magnitude higher with ~10-4-10-3 amino acid misincorporations per codon. Such errors become increasingly frequent with ageing, posing a sizable risk for the organism. In some instances, this leads to missing or misread stop codons, allowing translation to continue into the 3’UTRs of transcripts. Such C-terminal extensions may interfere with the folding of proteins, or worse, promote promiscuous interactions with other proteins, which in turn may disturb cellular processes and reduce overall fitness. Translation into the polyA-tail of transcripts leads to the activation of the ribosome quality control (RQC) complex, which clears both aberrant protein and mRNA.
However, in most cases, translation would be terminated at stop codons within the 3’UTR before the ribosome reaches the polyA-tail. Such readthrough events would therefore not be recognized by the RQC. While previous studies suggested that such readthrough products are recognized and efficiently cleared by cells, the underlying mechanism remained unclear. Given the decline in translational fidelity during ageing, this clearance pathway is expected to become increasingly important to release the burden on the proteostasis network. Using the nematode C. elegans as a model for ageing, we aimed to identify the quality control mechanisms mitigating translational readthrough and investigated the consequences of their failure during ageing.
Using this approach, we identified in C. elegans and human cells that readthrough proteins are cleared through a coupled, two-level quality control pathway involving the BAG6 chaperone complex and the ribosome collision-sensing protein GCN1. Readthrough proteins with hydrophobic C-terminal extensions are recognized by SGTA-BAG6 and ubiquitylated by RNF126 for proteasomal degradation. Additionally, cotranslational mRNA decay mediated by GCN1 and CCR4/NOT limits the accumulation of readthrough proteins. Selective ribosome profiling uncovered a general role of GCN1 in regulating translation dynamics when ribosomes encounter non-optimal codons, a feature of 3′UTR sequences. Dysfunction of GCN1 results in mRNA and proteome imbalance, increasingly affecting transmembrane proteins and collagens during ageing. These results define GCN1 as a key factor acting during translation in maintaining protein homeostasis
Selective and stepwise functionalization of the pyridazine scaffold by using thio-substituted pyridazine building blocks
Get PDFIn vitro Untersuchung der Wirkung verschiedener Sealermaterialien auf Zellen des oralen Systems im Hinblick auf Zytotoxizität und Entzündungsreaktion
Get PDFRegulatory mechanisms of the TRPM7 channel-kinase
Get PDFThe transient receptor potential cation channel, subfamily M, member 7 (TRPM7) is a bifunctional protein containing a transmembrane channel segment and a serine/threonine-protein kinase domain. The TRPM7 channel is selective to divalent cations. TRPM7 regulates many biological processes such as the organismal balance of Zn2+, Ca2+ and Mg2+, immunity, embryonic development and cell signalling. The widely accepted view is that cytosolic Mg2+ and Mg·ATP inhibit the TRPM7 channel to link the metabolic state of the cell to the uptake of divalent cations. More recently, independent studies focusing on the identification of native TRPM7 channel complexes revealed the association of TRPM7 with the metal transporters CNNM1-4. However, the mechanisms underlying TRPM7 sensitivity to intracellular Mg2+ and the regulatory role of CNNM1-4 remain poorly understood. Therefore, we used site-directed mutagenesis, patch-clamp measurements and other techniques to demonstrate that a regulatory Mg2+ binding site is formed by the side chains of N1097 in mouse TRPM7. Our results suggest a direct interaction between Mg2+ and N1097 leading to a stabilization of the TRPM7 in the closed conformation. In line with these findings, we showed that the sensitivity of TRPM7 to physiological concentrations of intracellular Mg2+ was selectively affected by an introduction of point mutation (N1097Q). In addition, our results indicate that the CNNM3 protein has no effect on the TRPM7 channel but rather negatively regulates the kinase activity of TRPM7. Thus, our study suggests new mechanistic insights into the regulatory characteristics of the kinase-coupled TRPM7 channel.Der Transient-Rezeptor-Potential-Kationenkanal, Unterfamilie M, Mitglied 7 (TRPM7) ist ein bifunktionelles Protein, das ein Transmembrankanalsegment umfasst, was mit einer Serin/Threonin-Proteinkinase-Domäne fusioniert ist. Der TRPM7-Kanal ist selektiv für Zn2+, Ca2+ und Mg2+. TRPM7 reguliert zahlreiche biologische Prozesse wie die Zn2+-, Ca2+- und Mg2+-Homöostase im Organismus, die Embryonalentwicklung, Immunreaktionen und Signaltransduktion. Eine weit verbreitete Ansicht ist, dass zytosolisches Mg2+ und Mg·ATP als negative Regulatoren des TRPM7-Kanals wirken, um die Aufnahme zweiwertiger Kationen auf den Stoffwechselzustand der Zelle anzupassen. Kürzlich haben unabhängige Studien, die sich auf die Identifizierung nativer TRPM7-Kanalkomplexe konzentrierten, die Verbindung von TRPM7 mit den Metalltransportern CNNM1-4 aufgezeigt. Die Mechanismen, die der Sensitivität von TRPM7 gegenüber intrazellulärem Mg2+ und der regulatorischen Rolle von CNNM1-4 zugrunde liegen, sind jedoch nach wie vor kaum verstanden. Daher haben wir eine Kombination aus ortsgerichteter Mutagenese, Patch-Clamp-Techniken, Western-Blotting und 3DProteinmodelierung eingesetzt, um zu zeigen, dass die Seitenketten von N1097 in TRPM7 in der Maus zwischen den Untereinheiten eine Mg2+-regulierende Stelle bilden. Unsere Ergebnisse legen nahe, dass Mg2+ direkt mit diesem Proteinsegment interagiert und dadurch TRPM7 im geschlossenen Zustand stabilisiert. In Übereinstimmung mit diesem Modell haben wir festgestellt, dass Punktmutationen in der Mg2+-Regulationsstelle (N1097Q und N1098Q) die Sensitivität von TRPM7 gegenüber physiologischen Konzentrationen von intrazellulärem Mg2+ aufhebt. Darüber hinaus deuten unsere Ergebnisse darauf hin, dass das CNNM3-Protein keine Auswirkungen auf den TRPM7-Kanal hat, sondern vielmehr die Kinaseaktivität von TRPM7 negativ reguliert. Unsere Studie liefert somit neue mechanistische Erkenntnisse über die regulatorischen Eigenschaften des Kinase-gekoppelten TRPM7-Kanals
